Researches On The Expression Of Hsa-mir-96in Bladder Urothelial Carcinoma And Its Biological Behavior Influence On Bladder Urothelial Carcinoma

According to discoveries from research on Epidemic epidemiology and Etiology, The incidence rate of bladder cancer ranks ninth of all human malignant tumors, as No.6among the male and behind No.10among the female. However, in China men's incidence of this disease ranks eighth among malignant tumors, while that of women behind twelfth. Bladder cancer can happen to people at any age, even children, but mainly the middle-aged. Many factors trigger bladder cancer, mostly including heredity and environment. Histologically, bladder cancer consists of papillary urothelial (transitional) cell carcinoma, squamous cell carcinoma gland carcinoma, and secondly rare small cell carcinoma, mixed type, carcinosarcoma and metastatic carcinoma. Among them urothelial cell carcinoma of the bladder accounts for above90%.MicroRNA (miRNA for short), a kind of small molecule of uncoded protein inherent in Eukaryotic cells, with a length of18-25nt, can cause degradation of target-mRNA or inhibit its translation through base pairing target-mRNA specificity, and thus achieve post-transcriptional gene expression regulation. It can exert its biological function by inhibitting downstream expression. MicroRNA can regulate the process of the proliferation and apoptosis of tumor cells, and participate in the process of the self-renewal and differentiation of cells. As a biomarker, it can be applied to cancer diagnosis, prognosis assessment and targeted therapy. Some miRNA molecules can also be used as protocarcinogenic genes or tumor-suppressor genes to regulate tumors.Reported by literature[1], the gene sequencing of the expression of miRNA in bladder urothelial carcinoma has discovered the high expression of hsa-mir-96in bladder urothelial carcinoma. Here ways of RT-PCR,Western Blot and so on are adopted to do preliminary research in molecular mechanism of hsa-mir-96growing in bladder urothelial cells, so as to forecast the further research in signaling pathways.Chapter1Detection of the expression of hsa-mir-96in bladder urothelial carcinomaObjective:To discuss and detect the expression and significance of hsa-mir-96in bladder urothelial carcinomaMethods:Detect the expression of hsa-mir-96in33cases of bladder urothelial carcinoma and9cases of normal bladder tissues near the carcinoma by Real-time PCR, and analyze the relationship between hsa-mir-96and the Pathology classification and clinical stages of bladder urothelial carcinoma.Results:The expression of hsa-mir-96in bladder urothelial carcinoma apparently exceeds that in normal bladder tissues near the carcinoma(P<0.01). We find the expression is closely related to the Pathology classification and clinical stages of bladder urothelial carcinoma.Conclusions:1. The expression of hsa-mir-96in bladder urothelial carcinoma is higher than that in normal bladder tissues near the carcinoma.2. There is an apparent positive correlation between the expression of hsa-mir-96in bladder urothelial carcinoma and that in normal bladder tissues near the carcinoma.3. There is a positive correlation between clinical stages of bladder urothelial carcinoma and the expression of hsa-mir-96.4. hsa-mir-96plays an important role in the process of occurrence and development of bladder urothelial carcinoma. Chapter2Effects of hsa-mir-96on the biological behavior of cell color of bladder cell carcinomaPart One Screening Tool Cells and Target GenesAccording to the expression of hsa-mir-96in bladder carcinoma strains, screen those cell strains with high expression as tool cells.Objective:To compare the expression of hsa-mir-96on bladder carcinoma strains T24and5637, and pick out the ones that can be used as tool cells for the cell level experiments. At the same time, we tested the expression of target genes IRS1, MAP4K1, MAP2K2, MAP3K3, MAP4K4on cancer cell strains, and select three of them whose expressions were relatively high for downstream experiments.Methods:We chose two strains of bladder carcinoma, T24and Bladder tumor cell line5637. Test the expression of hsa-mir-96on both of them with the method of Real-time PCR, determine the one whose expression degree is suitble for carrying out the interference experiment. And combine cell proliferation and differentiation characteristics, decide what can be used as tool cells for the cell level experiments, in addition, adopt semi-quantitative RT-PCR to test the relative content expression of target genes IRS1and MAP3K(MAP4K1, MAP2K2, MAP3K3, MAP4K4) on both cell strains. With the gene prediction softwares miRBase, TargetScan and Microrna to integrate the above target genes, we select three of them whose expressions are relatively high for downstream experiments.Results:The expression of hsa-mir-96on bladder carcinoma strains T24apparently exceeds that on Cell5637(P<0.05). In Cell T24and Cell5637, the relative R value of IRS1, MAP4K1, and MAP2K2is higher than that of MAP3K3and MAP4K4.(The relative R value=the relative IOD value of target gene/the relative IOD value of reference gene). Conclusion:1. There is significant difference between the expression of hsa-mir-96in bladder carcinoma strain T24and Cell5637. Therefore, cell T24is chosen as as tool cells for downstream experiments.2. In Cell T24and Cell5637, the R value of IRS1, MAP4K1, and MAP2K2is higher than that of MAP3K3and MAP4K4. Therefore, these3genes are chosen as the target gene of hsa-mir-96.3. According to the above results, genes IRS1, MAP4K1, and MAP2K2have higher expression in Cell T24. Therefore, both the3genes and Cell T24are chosen for downstream experiments.4. The validation on target genes suggests that the target gene gained by means of gene prediction softwares has false positive. Part Two Effects of hsa-mir-96on the cytobiological behavior of bladder carcinoma strain T24Objective:To explore the influence of hsa-mir-96on biological behaviors of the cell proliferation, apoptosis and invasive ability of bladder carcinoma strain T24.Methods:1. Construct hsa-mir-96inhibitors and negative clips of microRNA, transfect them into Cell T24, and confirm whether the transfection is successful through quantitative PCR detection2.Detect cell viability by means of MTT and draw the graph of the growth of cells.3. Detect the apoptosis of Cell T24by means of AV/PI Double Labeling Method FCM.4. Observe the influence of hsa-mir-96on the invasive ability of Cell T24through cell invasion assay.5. Sort the cells into3groups:Group A:Normally developed Cell T24.Group B:Negative control of microRNA.Group C:Inhibition of hsa-mir-96(Cell T24+clips of hsa-mir-96inhibitors).Results:1. Construct hsa-mir-96inhibitors and negative clips of microRNA, transfect them into Cell T24, and subsequent detection can be carried out. if the transfection rate is over80%.2. After transfection, the speed of Cell T24's growth is obviously slower than that of the controls, and the difference hsa statistical significance(P<0.01).3. After transfection, the apoptosis rate of Cell T24obviously increases compared with the controls(P<0.01). 4. It is observed through cell invasion assay that the invasive ability of Cell T24is weakened after transfection (P<0.01).Conclusions:1. hsa-mir-96inhibitors can restrain the growth of Cell T24.2. hsa-mir-96inhibitors can promote the apoptosis of Cell T24.3. The invasive ability of cells in Negative control of microRNA is reduced. Chapter3Researches on pathways of hsa-mir-96signal transductionObjective:According to the expression of hsa-mir-96target genes on Cell T24, we make preliminary study on the interaction relationship between hsa-mir-96and target genes, then do further study on the signal transduction pathways of hsa-mir-96in bladder urothelial carcinoma.Methods:1. Detect the expression of3genes (IRSI,MP4K1,MAP2K2) on Cell T24with the approach of Real-time PCR2. Detect the changes of protein content in the3genes with the approach of Western-blot.Results:1. The results of Real-time PCR shows that the relative contents of IRSI and MP4K1have obviously changed in3groups of cells, while that of MAP2K2hsa no distinct change.2. The results of Western-blot also proves that the protein expression of IRSI and MP4K1have obviously changed while the protein content of MAP2K2hsa no distinct change (P>0.05)Conclusions:1. Compared with those in normal control groups and negative control group, the expression of IRSI in the group of inhibitors is apparently different(P<0.01).2. Compared with those in normal control groups and negative control group, the expression of MP4K1in the group of inhibitors is apparently different (P<0.01).3. The relative protein content and expression of MAP2K2has no change in the3groups of cells, so it can't be used as target gene of hsa-mir-96. However, IRSI and MP4K1can serve as target gene of hsa-mir-96and they play an important role together with hsa-mir-96in bladder urothelial carcinoma.